Recently a large number of protein and glycoproteins have been identified that appear to mediate cellular proliferation and differentiation processes. See J. Immunol. 129, 2431-2436, (1982), Ihle, J. N. et al.; J. Immunol. Methods 42, 253-284, (1981) Stanley, E. R., et al.; J. Biol. Chem. 252, 5558-5564 (1977) Miyake, T. et al.; Proc. Natl. Acad. Sci. U.S.A. 81, 871-875 (1984) Stern, A. S. et al.; and, Adv. Protein Chem. 30, 251-451 (1976) Cuatrescasa, P.
Many of these proteins and glycoproteins are active in the 10.sup.-9 to 10.sup.-13 M range. This high potency has facilitated their detection and purification from biological fluids and conditioned media but at the same time has highlighted the need for more sensitive protein determination assays than those currently available. For example, a typical large scale purification of murine Interleukin-3, starting with 150 liters of conditioned medium, yields only 2-10 .mu.g of pure protein. (See Ihle, J. N. et al. above.) Thus, using either, the standard Bradford, Anal. Biochem. 72, 248-254, (1976) or Lowry et al. J. Biol. Chem. 193, 265-275 (1951) procedures, all of this pure material would have to be sacrificed in order to quantitate accurately the amount of protein present.
In 1976, Bradford successfully adapted the Coomassie protein staining procedure, initially developed for detecting proteins in SDS polyacrylamide gels, J. Biol. Chem. 244, 4406-4412 (1969) for quantitating protein levels in solution.
The applicant is aware of the following references which discuss methods of staining proteins in SDS-polyacrylamide gels utilizing the principle of silver binding.
Anal. Biochem. 98, 231-237 (1979) Switzer, III, R. C., et al.; Anal. Biochem. 105, 361-363 (1980) Oakley, B. R., et al.; Anal. Biochem. 110, 201-207 (1981) Merril, C. R., et al.; Electrophoresis 2, 135-141 (1981) Sammons, D. W. et al.; Anal. Biochem. 117, 307-310 (1981) Morrissey, J. H.; Anal. Biochem. 129, 227-287 (1983) Giulian, G. G. et al.; Anal. Biochem. 129, 490-496 (1983) Dion, A. S. et al.; Anal. Biochem. 131, 312-317 (1983) Schleicher, M., et al.; Anal. Biochem. 135, 409-415 (1983) Ohsawa, K., et al. PA0 Handbook of Biochemistry, Selected Data for Molecular Biology (Sober, H., ed.), Kirschenbaum, D. M. (1970) 2nd ed., pp. C/71-C/98, Chemical Rubber Company, Cleveland, Ohio. Anal. Biochem. 55, 166-192 (1973) Kirschembaum, D. M. The use of drained gel permeation beads for desalting protein solutions is described in Anal. Biochem. 55, 328-330 (1973) Neal M. W. et al.
Useful background references are: